The present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. More specifically, it relates to the reproduction of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco. The invention is especially suited for producing large numbers of clones of superior selections useful for reforestation.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are probably the most important commercial species of temperate Noah American timber trees. Similarly, Norway spruce (Picea abies (L.) Karst.) is probably the most important European softwood species. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted on cut-over or burned forest lands. For many years these seedling trees were grown using as naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees discovered in the forests. These trees were selected for such heritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducing some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
A brief review of some of the most important work relating to the present invention will follow. This is intended to be representative only and may not be fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full citations of the literature noted herein.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, potential genetic gain, and much lower probability of mutations. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage.
Earlier U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, and 5,236,841, herein incorporated by reference describe improved methods of conifer embryogenesis. These also include extensive reviews of the most closely related literature. In the methods described in all of these patents, advanced early stage embryos (or "late stage proembryos"), defined as totipotent embryonic structures estimated to have least about 100 mostly undifferentiated cells, are transferred to and further cultured in a cotyledonary embryo development medium containing abscisic acid (ABA) as an essential growth hormone. It appears to be highly desirable during this stage to gradually reduce the level of exogenous ABA so that little or none is ultimately present. Other growth hormones: e.g., gibberellins, may also be used at this time. The ultimate product of this culturing step is somatic embryos resembling natural zygotic embryos in morphology.
It is well accepted that plant tissue culture is a highly unpredictable science. Sondahl et al., in published European Patent Application 293,598, speak directly to this point.
"Since each plant species appears to possess a unique optimal set of media requirements, the successful preparation and regeneration of a new species cannot be necessarily inferred from the successful regimens applied to unrelated plant species." PA1 "Despite progress, our knowledge of embryogenesis is still fragmentary. At present we cannot yet define the conditions necessary for embryogenesis . . . " PA1 "It seems that the effects of activated charcoal are due to the removal of substances from the medium which promote unorganized growth, inhibit embryogenesis, root formation and elongation. One of these substances might be auxin . . . . " PA1 "The addition of activated charcoal to both liquid and semi-solid media is a recognized practice in plant tissue culture. . . . [T]he most certain effect of adding AC to media is that of lowering the levels of plant growth regulators and other organic substances."
This statement can be carried even farther. Rangaswamy (1986) notes that the potential for embryogenesis is even genotype specific within any given species.
Compositions of the media used to initiate embryogenesis and induce embryo maturation are critical to success, regardless of the species being propagated. In particular, the type and level of the nitrogen source in the media and the presence or absence, composition, level, and timing of availability of growth hormones have been the key to success. It is also these very factors, particularly the hormones, that have proved to be so unpredictable. As one example, Ammirato (1977), conducted a study examining the effects of zeatin (a cytokinin), ABA, and gibberellic acid (GA.sub.3) on the yield and morphology of caraway (Carum carvi) somatic embryos. These hormones were present singly and in all possible combinations in the media used for the later stages of embryo development. He concluded that a change in level or presence/absence of any one of the hormones caused a ripple effect felt throughout the system due to unpredictable interactions between the various hormones. Lakshmi Sita (1985) summarizes her earlier work and that of others in promoting embryogenesis of sandalwood (Santalum sp.). Gibberellic Acid was found to be useful in inducing embryogenesis using shoot explants in either solid or liquid suspension cultures. Despite her success, which included successful production of convened plants, she again points to the lack of predictability of embryogenesis.
The same problem is again discussed by Evans (1984) who notes that growth hormones which affect the same process can either act independently or may interact in some fashion.
In general, as far as coniferous species are concerned, it appears that at least one exogenous auxin and usually a cytokinin are necessary hormones in a medium for the initiation of embryogenesis. To the present time it has also appeared necessary to include abscisic acid in the media for development of early stage embryos into cotyledonary stage embryos capable of germination and growth into plants. Douglas-fir, the subject species of the present invention, is known to have an embryogeny quite different from most other coniferous species. In the seed formation of most other conifers multiple embryos are present. Ultimately as the seed matures one embryo will become dominant and the others will either atrophy or be inactivated. However, in Douglas-fir only a single embryo is usually seen. This is discussed in some detail by Singh (1978). In actuality, Douglas-fir does appear to experience polyembryony very early in seed formation. However, all but one embryo is quickly suppressed. While the exact cause of this phenomenon is not known for sure, it appears to be related to a high level of endogenous abscisic acid (ABA) in the seed. Douglas-fir differs from other species as well during tissue culture. After initiation of early stage embryos, many of the embryos tend to form clumps of several individual embryos. Following the maintenance and multiplication stage, in the past these clumps have been separated into individual embryos by repeated transfers in liquid culture using media of relatively low osmolality. These "singulation" media initially required a relatively high level of exogenous abscisic acid which was reduced stepwise fashion in succeeding cultures. Ultimately, a very sharply raised osmolality and a relatively low level of ABA is needed in the cotyledonary embryo development culture. Even that low level of ABA is preferably continuously reduced by the presence of a hormone adsorbent such as activated charcoal. U.S. Pat. Nos. 5,034,326, 5,036,007, and 5,236,841 describe this process in considerable detail.
The use of activated charcoal as an additive in tissue culture has a long history. Fridborg and Eriksson (1975), citing even earlier workers, note the benefits of activated charcoal as used in their specific media. They comment as follows:
More recently this statement has been made more emphatic. In a 1990 paper Ebert and Taylor stated:
Most of the reported work using charcoal containing media has been on the various angiosperm species. As one example, Johansson (1983) found activated charcoal to strongly promote embryogenesis in anther cultures of Anemone canadensis. However, it is well established that a procedure that works well on one group of very closely related plants may be totally ineffective on unrelated plants. This is especially true when comparing plants as distantly related as those in different botanic Orders or Families. Success has been poor enough when using similar procedures between different members of the same genus.
In most tissue culture procedures, especially those involving conifers, activated charcoal has been used at the initiation stage of embryogenesis or for germination of cotyledonary stage embryos. On much rarer occasions it has been used in conifer embryogenesis in a single short duration subculture between the initiation and maintenance stages of culture. The three patents just listed above provide an exception where activated charcoal is used in the cotyledonary embryo development stage of culture to continuously reduce the level of abscisic acid in the medium.
Becwar, Nagmani, and Wann (1990), in work on loblolly pine, used activated charcoal in one of their initiation media and one maintenance medium. It was not used at the cotyledonary embryo development stage.
In a paper concerned mostly with initiation of embryogenic cultures of Norway spruce, Hakman, Fowke, von Arnold, and Eriksson (1985) noted that when cultured on a medium containing activated charcoal, embryos developed visible cotyledons and roots but this was only possible after the embryos had reached a certain stage of maturity. Since the exact procedures used by these workers was vague, it is unclear exactly when and how charcoal was used and whether it was or was not used in a cotyledonary embryo development stage.
In a paper published in 1985, Gupta and Durzan used activated charcoal in an elongation medium for sugar pine (Pinus lambertiana) buds in an organogenesis culture. These same authors in 1986 and 1987 papers described media containing activated charcoal in media for germination of somatic embryos of sugar pine and loblolly pine. Durzan and Gupta (1987) used the same treatment in the germination stage of Douglas-fir.
More recently, Becwar, Noland, and Wann (1987) and Verhagen and Wann (1989), working with Norway spruce, subcultured newly initiated embryogenic callus to a medium with 1% activated charcoal and lacking growth regulators for one week, followed by transfer to a maintenance medium consisting of their basal medium with 1 .mu.m each of indolebutyric acid (IBA) and abscisic acid (ABA). Charcoal was not used elsewhere in their culturing procedure. A similar protocol was used by Uddin as described in U.S. Pat. No. 5,187,092.
Even with the knowledge of these reported instances of use of activated charcoal in conifer tissue culture media, it has not been at all apparent when it might be beneficial or detrimental nor has it been consistent in its expected results.
Techniques to promote embryogenesis of various members of several conifer genera are now well established. Research emphasis is now shifting to development of ways to scale up laboratory knowledge and techniques so that the process may become field operational on large scale. Yet many problems of a relatively fundamental nature still remain to be solved. One of these is improving somatic embryo quality and vigor and of reducing the cost. This is necessary so that germination to hardy plantlets and ultimate conversion to growing trees can be achieved at much higher percentages and much more economically than has heretofore been possible.